When naive T cells encounter foreign antigen along with proper co-stimulation and cytokines, they undergo extensive clonal expansion and differentiate into specific lineages. In mammals, this type of proliferation is fairly unique to cels of the adaptive immune system and requires a considerable expenditure of energy and cellular resources. While research has often focused on the roles of cytokines, antigenic signals, and co- stimulation in guiding T cell responses, recent data indicate that, at a fundamental level, it s cellular metabolism that regulates T cell function and therefore influences the final outcome of the adaptive immune response. Indeed, a role for the metabolic pathways in T cell activation is beginning to be appreciated, but little is known about their involvement in the differentiation of regulatory T (Treg) cells, a central cell type in maintaining immune tolerance and inhibition of autoimmune and inflammatory diseases. We have found that S1P1, a G protein-coupled receptor (GPCR) for the bioactive lipid sphingosine 1-phosphate (S1P), inhibits differentiation of Treg cells. Moreover, S1P1 activates mTOR, a central regulator of protein translation, cellular metabolism and various other processes. Although activation of mTOR has been shown to restrain Treg differentiation, the mechanism involved remains unknown because of the pleiotropic functions of mTOR. We propose a novel concept that a low metabolic activity is actively regulated to allow the differentiation of Treg cells, and our long-term goal is to identif function and regulation of the metabolic machinery in T cell differentiation. In this exploratory/developmental grant application, we hypothesize that the S1P1-mTOR axis serves as a metabolic checkpoint to link cellular metabolism and negative control of Treg differentiation. We will test our hypothesis by establishing the signaling mechanisms of S1P1 and mTOR in Treg differentiation, and determining whether the metabolic machinery activated by S1P1 and mTOR controls Treg differentiation. These studies provide a series of tests and explorations of the new concept we propose, and hold the potential to greatly advance our understanding of the fundamental processes of cell metabolism and T cell differentiation. Moreover, given that S1P1 and mTOR are important therapeutic targets for transplant rejection and autoimmune and inflammatory diseases, our studies can be translated into innovative strategies to treat these immune-mediated diseases. PUBLIC HEALTH RELEVANCE: Regulatory T cells play a central role in the maintenance of immune tolerance to self tissues and in the down- modulation of the immune responses to infection and cancer. Abnormal functions of regulatory T cells can lead to autoimmune diseases as well as immunodeficiency. Therefore, a better understanding of the molecules and pathways involved in regulatory T cells is essential for future efforts to prevent and treat these immune- mediated diseases.